The cellular response to extracellular vesicles is dependent on their cell source and dose

Extracellular vesicles (EV) have been established to play important roles in cell-cell communication and have shown promise as therapeutic agents. However, we still lack a basic understanding of how cells respond upon exposure to EVs from different cell sources at various doses. Thus, we treated fibroblasts with EVs from twelve different cell sources at doses between 20 and 200,000 per cell, analyzed their transcriptional effects, and functionally confirmed the findings in various cell types in vitro, and in vivo using single-cell RNA-sequencing. Unbiased global analysis revealed EV dose to have a more significant effect than cell source, such that high doses downregulated exocytosis and upregulated lysozomal activity. However, EV cell source-specific responses were observed at low doses, and these reflected the activities of the EV’s source cells. Finally, we assessed EV-derived transcript abundance and found that immune cell-derived EVs were most associated with recipient cells. Toget...,
EV uptake experiments

Five thousand human primary fibroblasts (NIGMS Human Genetic Cell Repository #GM08402) were seeded into flat bottom 96-well plates and incubated with 90ul full medium with 10ul PBS-HAT buffer containing 1e5, 1e6, 1e7, 1e8 or 1e9 EVs for 24 hours. Cells were then washed twice with PBS and analyzed. 

RNA-sequencing

Cell or EV RNA was extracted (32) and precipitated as previously described (3) by incubating 500ul TRI Reagent (Sigma), adding 100ul chloroform, and shaking vigorously. After a 15 minute incubation, samples were centrifuged at 12,000 x g for 15 minutes at 4C and 300ul of aqueous phase was mixed with 300ul isopropanol, 30ul of 3M sodium acetate, and 1ul Pellet Paint (Merck) and incubated overnight at –20C. The next morning, samples were centrifuged at 20,000 x g for 30 minutes at 4C, the pellets were washed two times with 700ul of 70% ethanol, before drying and resuspending in 15uL elution buffer (Qiagen). RNA concentrations were measured using Qubit..., R and R Studio, with required packages listed in the scripts.